Potential cross-species transmission of highly pathogenic avian influenza H5 subtype (HPAI H5) viruses to humans calls for the development of H5-specific and universal influenza vaccines

In recent years, highly pathogenic avian influenza H5 subtype (HPAI H5) viruses have been prevalent around the world in both avian and mammalian species, causing serious economic losses to farmers. HPAI H5 infections of zoonotic origin also pose a threat to human health. Upon evaluating the global distribution of HPAI H5 viruses from 2019 to 2022, we found that the dominant strain of HPAI H5 rapidly changed from H5N8 to H5N1. A comparison of HA sequences from human- and avian-derived HPAI H5 viruses indicated high homology within the same subtype of viruses. Moreover, amino acid residues 137A, 192I, and 193R in the receptor-binding domain of HA1 were the key mutation sites for human infection in the current HPAI H5 subtype viruses. The recent rapid transmission of H5N1 HPAI in minks may result in the further evolution of the virus in mammals, thereby causing cross-species transmission to humans in the near future. This potential cross-species transmission calls for the development of an H5-specific influenza vaccine, as well as a universal influenza vaccine able to provide protection against a broad range of influenza strains.


Introduction
Avian influenza is an infectious disease that affects poultry and wildfowl. It is caused by highly pathogenic avian influenza (HPAI) or low pathogenic avian influenza (LPAI) viruses, which belong to the Orthomyxoviridae family and have a single-stranded negative-sense RNA genome. Avian influenza viruses (AIVs) are mainly classified on the basis of their surface proteins, hemagglutinin (HA) and neuraminidase (NA). HA protein on the surface of the virion, the main antigenic site in vaccine design, causes erythrocyte agglutination in vitro and in vivo 1 . Over the years, outbreaks of HPAI H5 subtype viruses in poultry have caused huge economic losses to the farming industry. In 2022, more than 25 million poultry and wild birds were infected with HPAI H5 worldwide, resulting in 5.28 million deaths (https:// wahis.woah.org/). Recently, HPAI H5 has caused more sporadic cases, or even outbreaks, in mammals, including minks, otters, foxes, and sea lions [2][3][4] . With possible further mutations in avian and mammalian species, HPAI H5 has a strong potential to cause human infection and trigger a global pandemic. Therefore, it is essential to develop an H5specific vaccine, as well as a universal influenza vaccine, to fully cover a broad range of influenza strains.

Global distribution of HPAI H5 viruses
H5N1 was the first strain isolated among the HPAI H5 viruses in Scotland in 1959, and it was shown to infect a variety of avians 5 . In 1997, HPAI H5N1 (Gs/GD/96) emerged in China and it was first confirmed to infect humans 6 . In 2000, H5N1 broke out among poultry in several countries, including the Netherlands, Vietnam, Indonesia, and Thailand 7 . A few years later (after 2005), H5N1 further spread to poultry in Europe and Africa 8,9 . Owing to homologous recombination among influenza strains in poultry, other non-N1 recombinant AIVs strains, such as H5N2, H5N6, and H5N8, have emerged in many countries. To classify H5 subtype AIVs, the HA gene was selected by the WHO/OIE/FAO H5N1 Evolution Working Group to divide AIVs into diffident clades based on the similarity of HA nucleic acid sequences. Each distinct clade was determined to have an average distance > 1.5% from other clades 10 . From 2013 to 2019, HPAI viruses of subclades 2.3.2.1 and 2.3.4.4 began to spread around the world [11][12][13][14][15][16] . HPAI H5 subclade 2.3.4.4 was first detected in domestic ducks in China 17,18 and was further divided into 8 subclades, 2.3.4.4a to 2.3.4.4h 19 .
From 2019 to 2022, HPAI H5 viruses have been circulating among avian populations in Europe, Africa, and Asia [20][21][22][23][24] , resulting in a significant increase in global avian cases from 0.343 to 25.19 million (Fig. 1). Europe has become the primary site of spread accounting for 82.7% of avian cases and 43.9% of deaths globally in 2022 (Fig. 2a). Notably, the main HPAI subtype virus causing global epidemics gradually changed from H5N8 to H5N1 between 2019 and 2022 (Fig. 2b). For example, the epidemic of HPAI viruses in Europe was dominated by H5N8 from 2019 to 2021. However, it changed to H5N1 in 2022.
During this year, infections and mortality rates caused by H5N1 accounted for~99.9% among all HPAI H5 viruses in the same period (Fig. 2c). Since 2019, the H5N1 subtype has been dominant in Africa and the Americas, accounting for more than 99.9% of cases (Fig. 2d, e). Similar to Europe, H5N8 was the main HPAI subtype in Asia from 2019 to 2021, but it also changed to H5N1 in 2022. In 2022, the H5N1 subtype accounted for 67.4% of infections and 76.3% of mortalities among all H5 subtypes in Asia (Fig. 2f).

Human infections with HPAI H5 viruses
The current global epidemic of HPAI H5 mainly involves three subtypes: H5N1, H5N8, and H5N6. Indeed, the widespread epidemic of AIVs among wild birds increases the risk of infection for poultry and other avians. However, it is generally believed that human infection with HPAI H5 viruses is closely related to outbreaks in poultry and wild birds, and according to the WHO, 864 human cases of H5N1 infection have been reported worldwide, resulting in 456 deaths from 2014 to 2021 (Fig. 3a). It was previously believed that only cumulative mutations of AIVs in avians could lead to spillover, causing mammalian (or human) infections and deaths. Currently, no evidence of HPAI H5 transmission has been observed among mammals. However, H5N1 was recently detected in mink farms in the United States and Spain, and more than 50,000 mink were killed to prevent further spread [2][3][4] . These events provide strong evidence that HPAI H5 viruses can spread rapidly among mammals and that minks may serve as a potential intermediate host to increase the possibility of the H5N1 epidemic in humans. In fact, human infections caused by the HPAI H5 viruses have recently been reported in Ecuador, Cambodia, and Chile (https://www.who.int/emergencies/disease-outbreak-news/ item/2023-DON434; https://www.cdc.gov/flu/avianflu/ spotlights/2022-2023/chile-first-case-h5n1-addendum.htm).
More concerning evidence has been reported on human infection with the H5N6 subtype virus. From 2014 to 2022, 87 human cases of H5N6 infection were reported (86 in China and one in Laos), with most infections reported in 2021 and 2022 (Fig. 3b). The number of H5N6 infection cases in 2021 and 2022 accounted for 67% of the total number of infections from 2014 to 2022, suggesting that the Chinese government should strengthen protective measures to prevent further spread of the H5N6 virus. Additionally, the first case of H5N8 infection in humans was reported in Russia in 2020 25 . The significant increase in human cases of H5N6 and the emergence of a new human case of H5N8 are alarming signs for human safety.
Analysis of HPAI H5 HA sequences and assessment of the risk for human infection based on the infection data from 2019 to 2022 In the process of viral infection, HA binds to sialic acid receptors on the cell surface and mediates the fusion of the . c-f Continent-specific distribution of each subtype of HPAI H5. Raw data for avian cases and deaths were taken from WAHIS (https://wahis.woah.org/#/dashboards/qd-dashboard). Data are included up to January 2023. viral membrane and the host endosomal membrane to deliver viral nucleic acid into the cytoplasm of host cells, thereby playing a key role in the process of infection. HA protein is hydrolyzed to produce HA1 and HA2. HA1 binds to cell receptors via the receptor-binding domain (RBD) and is prone to mutations, while HA2 mediates the membrane fusion process and is relatively conserved. The HA1 of AIVs binds to the α-2,3-sialic acid receptors in avian species, while it binds to the α-2,6-sialic acid receptors in humans. The difference in receptor usage partly prevents the transmission of AIVs from birds to humans. Therefore, we compared HA sequence, HA1 sequence, and RBD key sites of HPAI H5 viruses isolated from avians and humans in recent years to assess the potential risk of human infection.
RBD is located in the head of HA1 and contains 190helix, 130-loop, 150-loop, 220-loop, and other amino acid residues 26,34,35 (Fig. 5). Yang et al. 36 found that the introduction of S137A and T192I mutations in the RBD of A/Thailand/KAN 1/2004 endowed this Avian strain with the ability to bind with α-2,6-sialic acid receptors present in humans. In our selected sequences, the 137A and 192I sites were found to be present in both human-and avianderived H5N8 and H5N6 strains, indicating that they are key sites for cross-species transmission. In addition, although 192T exists in A/Astrakhan/3212/2020 (H5N8) and A/Fujian-Sanyuan/21099/2017 (H5N6), these strains still retain the ability to infect humans. This indicates that a single-site mutation (S137A/T) may also change the receptor-binding ability of AIVs. It has been reported that some mutation sites, such as the K193R mutation in the A/Vietnam/1203/2004 strain 37 , the Q196H mutation in the A/duck/Egypt/ D1Br12/2007 strain 38 , and the Q226L, S227N, and G228S mutations in the A/Indonesia/05/ 2005 strain 39 , can enhance the ability of stains to utilize the α-2,6-sialic acid receptor. In our selected sequences, the 193R site is present in both human-and avian-derived H5N1 strains, indicating that these H5N1 strains may have already gained the ability to utilize α-2,6-sialic acid receptors.

Antiviral therapy for influenza
Some small-molecule compounds have been developed for the treatment of influenza viruses. These compounds target various stages of the viral life cycle, e.g., virus adsorption, fusion, nucleic acid release, nucleic acid replication, and virus budding. HA protein inhibitors block virus adsorption or fusion, which can be divided into HA1 and HA2 inhibitors. HA1 inhibitors, such as Dextran sulfate and DSA181 40 , block the binding of HA1 to receptors on the cell surface. Meanwhile, HA2 inhibitors like arbidol 41 and BMY-27709 42 block virus entry by preventing HA2-mediated membrane fusion. In addition, Basu et al. identified two small-molecule compounds, MBX2329 and MBX2546, which were able to bind to the stem region of the HA trimer and inhibit HA-mediated fusion 43 . The fusion process of the influenza virus also depends on endosomal acidification and a series of host enzymes, like proteases. Therefore, inhibitors of these host enzymes have also been developed as anti-influenza drugs, such as bafilomycin A1 44 and aprotinin 45 . After membrane fusion, viral RNA enters the host cell through the M2 ion channel. M2 inhibitors like amantadine and rimantadine, which block ion channel activity, were developed to prevent the release of the viral genome into the cytoplasm. M2 inhibitors are effective for the influenza A virus but not for the influenza B virus because of its lack of M2 protein. It has been reported that S31N   Table 2 Comparison of key amino acid sites in the RBD region of HPAI H5 viruses.   55 ).
In addition, some monoclonal antibodies (Mab) have been developed and are highly anticipated for postexposure prophylaxis and clinical treatment. For example, a novel humanized Mab 8A 56 neutralized H5N1 by binding to two types of epitopes on HA. Li et al. described a chimeric Mab, termed C12H5, which could neutralize representative strains of H1N1 circulating from 1991 to the present; it could even cross-neutralize H5N1 57 . It has been reported that neutralizing antibodies against HA, isolated from volunteers vaccinated with seasonal influenza vaccines, could protect mice from H1N1 and H3N2 viruses in vivo 58 . Recently, the FDA confirmed that humanized polyclonal antibody SAB-176 could recognize multiple epitopes and provide protection against multiple influenza virus strains (https://ir.sab.bio/static-files/ b332c893-5795-4d05-af96-a9ebcd917f24). A phase 2b clinical trial is about to be launched in patients with highrisk severe diseases. Some polypeptide drugs, such as EBpeptide 59 , iHA 60 , FluPep 61 , NDFRSKT 62 , P1 63 , and P9R 64 , have also been developed against influenza viruses. However, the accumulation of mutations in AIVs still increases the probability of immune evasion 65,66 . Therefore, updating existing antiviral drugs cannot keep pace with the continuous variation of AIVs. This calls for new antiviral strategies, such as drugs and therapeutic Mabs targeting more conserved viral epitopes or cytokines, or immunomodulatory drugs, in response to emerging strains with epidemic potential 43,67 .

Development of H5-specific influenza vaccines
Currently, the main types of avian influenza vaccines include inactivated recombinant vaccines, subunit vaccines, viral vector vaccines, and DNA vaccines. Inactivated vaccines were previously the primary means of preventing influenza and were mainly prepared from low pathogenicity strains isolated from farms 68 . However, traditional inactivated vaccines are not conducive to vaccine production owing to such defects as dependence on Table 2 continued  69

Development of universal influenza vaccines
As AIV is a single-stranded RNA virus, its nucleic acid sequence is prone to mutation, thereby reducing the protective efficacy of the vaccine over time. Although it is possible to predict the next dominant strain for vaccine strain selection, production, and distribution, the circulating strain may further mutate, resulting in a decrease in vaccine protection efficiency. Therefore, it is necessary to develop universal influenza vaccines that target more conservative epitopes to counter potential antigenic drift or shift in AIVs. Accordingly, scientists have focused on several common targets for the development of universal influenza vaccines, including the conserved stalk domain of HA protein, the conserved regions of NA protein, the ectodomain of M2 ion channel (M2e), and the internal proteins, nucleoprotein (NP) and matrix protein 1 (M1). The aim is to expand existing immune memory response by multiple immunizations in order to produce the widest range of protective antibodies against different subtypes of influenza virus 72 .
Effective humoral heterosubtypic immunity is rare, mainly based on antibodies targeting the HA stalk domain 73,74 . As mentioned, the RBD region in the head of HA protein is prone to mutations leading to viral immune evasion, while the HA stalk domain is rarely exposed to neutralizing antibodies, thus facing less selection pressure from the host immune system. As a result, the HA stalk domain is highly conserved in AIVs, making it an attractive target for universal vaccine design. A strategy for inducing high levels of stalk-reactive antibodies is based on chimeric HAs (cHAs), which combine exogenous head domains with conserved stalk domains. The cHAs with different head domains have been used in sequential vaccination programs to break the immunodominance of the head domain of HA and induce high titers of stalk-reactive antibodies 75 . However, the vaccine targeting the conserved stalk domain of HA can only produce cross-protection that occurs between strains within the same subtype or multiple subtypes of the same group, making it difficult to induce broadly reactive antibodies against influenza viruses across different groups. However, the construction of chimeric HA stalk domain with other conserved antigens, such as M2e, could improve cross-protection against multiple AIVs from different groups and improve the broad protection of universal influenza vaccines 76 . Chen et al. 77 have reported that influenza virus infection induces high titers of NA-reactive antibodies, which effectively inhibit the enzymatic activity of NA and provide robust prophylactic protection against avian H5N1 viruses in vivo. This observation suggests that some conserved regions in NA recognized by NA-reactive antibodies could be incorporated into influenza vaccines to elicit durable and broad protection against divergent influenza strains.
Emerging vaccine platforms can help trigger a better immune response than that induced by traditional influenza vaccines. For example, virus-like particles (VLPs) can present natural conformational antigens, stimulate the immune system through a virus-like pathway, and efficiently induce immune protection. An H5N1 VLP-based vaccine, designed with computationally optimized broadly cross-reactive antigen (COBRA), elicits broadly reactive antibodies in mice and ferrets. Therefore, this strategy is potentially paradigm-shifting for H5 universal influenza vaccines 78 . A candidate universal influenza vaccine, which uses M2e-based VLP to present M2e, has been shown to protect mice from homosubtypic and heterosubtypic AIVs 79 . In addition, nanoparticle platforms have been used to develop universal influenza vaccines owing to their dominance in expressing antigens at high densities and providing adjuvant-like functions. For example, the OVX836 vaccine is based on oligomerized nanoparticles (NPs) that can induce humoral and cellular immunity in mice and ferrets, thereby providing protection against influenza A and B 80,81 . A 'mosaic' quadrivalent influenza vaccine based on two-component nanoparticle immunogens not only showed better protective antibody response than the 2017-2018 quadrivalent influenza vaccine (QIV) but also triggered heterosubtypic antibody response and protective immunity in several animal models 82 . Viral vector-based vaccines can be delivered through both systemic or mucosal routes to trigger strong humoral and cellular immunity. An adenovirus vector-based H5N1 conserved multi-epitope influenza vaccine showed broad immune protection against H5, H7, and H9 influenza viruses in mice 83 86 developed an mRNA-LNP vaccine encoding HA from 20 known influenza A and B virus subtypes, and it triggered high levels of cross-reactivity and subtypespecific antibodies in mice and ferrets. This is a new antigen design concept for developing a universal influenza vaccine.
In addition, new vaccine adjuvants support ideas for universal vaccine design. Appropriate vaccine adjuvants can improve immunogenicity, regulate immune response types, and even enhance the universality of vaccine protection 87,88 . Only 6 new adjuvants have been approved by the FDA in the past century, including MF59, AS04, AS03, AS01, CpG1018, and Matrix-M adjuvants for emergency use in COVID-19. MF59 and AS03 have improved the protective efficiency of influenza vaccines 73,89 . More prominently, the quadrivalent influenza nanoparticle vaccine (qNIV) with Matrix-M has been shown to enhance antigen presentation, expand the antibody epitope library, boost cross-neutralizing antibody responses, and improve the induction of potent CD4 + and CD8 + T cell responses in a variety of cells 90 . It has now successfully completed key phase III trials. Compared with adjuvant-free vaccines, influenza vaccines with adjuvants have shown higher immunogenicity and effects on heterologous strains. The 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is an effective natural agonist of stimulator of interferon genes (STING), which induces type I interferon (IFN-I) response and proinflammatory cytokine production by activating STING 91,92 . Wang et al. 93 demonstrated intranasal immunization with the PS-cGAMP-adjuvanted inactivated H1N1 vaccine, which triggered a strong protective effect against different subtypes of influenza (H3N2, H5N1, and H7N9) in mice. During the COVID-19 pandemic, the emerging non-nucleotide small-molecule STING agonist CF501 showed higher protective efficacy compared to the cGAMP-adjuvanted vaccine, suggesting that CF501 can also be used as an adjuvant to boost the original vaccine for effective, extensive and long-term immune protection 94 .
In this article, we have analyzed the global epidemic of HPAI H5 and revealed that the number of infections has risen significantly in recent years. Furthermore, it has been observed that the dominant HPAI virus worldwide rapidly changed from H5N8 to H5N1 in 2022. According to the sequence alignment analysis of HA1, we found that the HA1 sequences of strains isolated from avians and humans were highly homologous or even identical, suggesting that the existing AIVs strains circulating in birds may infect humans. Amino acids 137A, 192I, and 193R in the RBD of HA are key sites that exist in both avian and human source sequences. These sites enable the current HPAI H5 strains to bind α-2,6-sialic acid receptors in humans, indicating that the mutated HPAI H5 viruses may have jumped from birds to mammals and that such spillover may cause human infection.
It should be mentioned that receptor affinity is not the only factor affecting the transmission of AIVs in humans. In the process of viral infection, HA mediates membrane fusion between the virus and host cells 95 . Next, nucleic acid is released with the assistance of the M protein and enters the nucleus to complete viral replication in the presence of viral polymerases PA, PB1, and PB2. Finally, the progeny virus is released from infected cells with the assistance of the NA protein. Many HPAI H5 viruses can enter host cells, but they cannot replicate successfully owing to the difference in amino acids at position 627 of PB2 protein, namely glutamic acid in AIVs and lysine in human influenza virus 96 . Hence, mutations in the RBD domain may only affect receptor binding and cell entry of AIVs, while replication efficiency of the virus in cells must be assisted by other viral proteins, such as PA, PB1, PB2, and NA, to gain successful cross-species transmission 97 . Therefore, mutations in these proteins and homologous recombination between strains deserve more attention.
Nowadays, the HPAI H5 virus belonging to the 2.3.4.4b subclade is widespread among wild birds and poultry worldwide, resulting in significant economic losses. The prevention and control strategy for the HPAI H5 virus in Europe and North America mainly relies on culling, while the strategy in China is "vaccine and culling". The latter strategy did reduce HPAI H5 virus infections in avians in China (Fig. 1a) 25 . In addition, after vaccination of the H5/ H7 vaccine in poultry, the isolation of H7N9 strains in China decreased by 93.3%, which largely prevented the prevalence of H7N9 among poultry 98 . The transmission modes of HPAI H5 among wild birds, poultry, and mammals also deserve more attention. Wildfowl is the natural host of the HPAI H5 virus, and the virus usually replicates in their intestines and respiratory tract. Nine major routes have been identified for migration across the world, increasing the likelihood of AIV infection in poultry and mammals 99,100 . Therefore, understanding the temporospatial characteristics and as well as environmental factors of HPAI H5 outbreaks is helpful for establishing an effective prevention and control system 101 . It is widely accepted that AIVs only infect mammals through avian transmission and that no reports have so far indicated its spread among mammals. Nonetheless, the recent spread of the H5N1 virus in mink has sounded the alarm for human safety [2][3][4] . Prevention should be emphasized in virus-susceptible areas, and measures should be taken to reduce human exposure to birds and mammals in order to minimize the risk of zoonotic infections. Protective measures and preventive vaccination should be taken seriously for populations susceptible to occupational hazards. Finally, real-time virus monitoring and rapid data sharing are crucial for assessing the risk of cross-species transmission of HPAI H5 and implementing effective prevention and control measures. The antigenic drift of the current epidemic strains should be monitored, and it should be determined whether the existing vaccines still have protective effects. Furthermore, H5-specific vaccines need to be developed, and the team led by Hualan Chen in China, whose work was noted above, serves as a model in this regard 71,102,103 .
While small-molecule compounds, peptides, and antibodies have been developed for influenza antiviral therapy, the constant mutation of the virus and its ability to evade immune response confound these efforts. Therefore, drugs and vaccines must be regularly updated to address the emergence of new strains. In response, scientists are trying different methods to develop universal vaccines against multiple subtypes of influenza viruses. HA is the main immunogen for vaccine design and mainly induces antibodies against the RBD region at the spherical head of HA, which is also highly prone to mutation. However, some cross-protective antibodies against highly conserved HA stalk may also be induced 104 . Emerging vaccine platforms and new vaccine adjuvants also provide pathways toward improving vaccine efficacy. Although not emphasized in this review, the potential of crossreactive T cell-based responses for influenza vaccine design cannot be ignored. Currently, avian influenza vaccines are mandatory for poultry immunization. However, they are not included in routine human immunization but are only used as a preventive vaccination strategy during emergencies. HPAI H5 viruses are circulating in birds and have even caused outbreaks in mammals in recent years, thus raising concerns about HPAI H5 infections in humans. Heterologous prime-boost immunization strategies against H5N1 could induce broader cross-clade antibody responses. It is also worth considering priming with a universal vaccine and boosting with a specific vaccine against the current pandemic strain.